1. Field of the Invention
This invention relates to the diagnosis of abnormal GH activity or general pathological activity in brown adipose tissue.
2. Description of the Background Art
Brown Adipose Tissue:
Brown adipose tissue (BAT) is known as a major site of heat production or thermogenesis where it normally consumes fat derived from white adipose tissues (WAT). Brown adipocytes generally reside in capillary beds and are abundant in cytochromes and proteins, particularly uncoupling protein-1 (UCP1). A BAT depot is located in the interscapular space in rodents, but exists in the abdominal, neck, and upper back areas in human neonates. In contrast to rodents, BAT gradually disappears as children grow. Recent studies demonstrated that brown adipocytes can also be found in rat and baboon WAT during cold stress (Cousin et al. 1992 and Viguerie-Bascands et al. 1996).
Growth Hormones:
The growth hormones are vertebrate proteins with about 191 amino acid residues, the number varying from species to species. There are four cysteine residues, and two disulfide bridges. The 3D-structure of porcine GH is known; it is composed of four major antiparallel alpha-helices, at residues 7–34, 75–87, 106–127 and 152–183.
The 3D structure of the hGH:hGH receptor complex is also known. Each molecule of hGH binds two molecules of the receptor. hGH binds to two binding sites on hGH receptor. Helix 4, the loop residues 54–74, and, to a lesser extent, helix 1, mediate binding to binding site 1. Helix 3 mediates binding to binding site 2.
See generally Harvey, et al., Growth Hormone (CRC Press:1995). GH is synthesized and secreted by the somatotrophic and somatomammotrophic cells of the lateral anterior pituitary. The control of GH production and secretion is complex, but is mainly under the influence of growth hormone releasing hormone (GHRH) and somatostatin, which stimulate and inhibit it, respectively. The shifting balance between these regulatory agents is responsible for the pulsatile nature of GH secretion, with normal human concentrations ranging from a baseline value <1 μg/L to peaks of 25–50 μg/L. Glucocorticoids and thyroid hormones, and various carbohydrates, amino acids, fatty acids and other biomolecules, are also known to directly or indirectly regulate GH secretion.
Most GH is secreted at night, during deep sleep, but some is secreted in response to exercise and other forms of physical stress. About 500 μg/m2 body surface area are secreted by women, and 350 by men. GH secretion rates are highest in adolescents and lowest in the elderly. GH has a plasma half-life of about 20–25 min. and is cleared at a rate of 100–150 ml/m2 body surface area.
Metabolic and Clinical Effects of Growth Hormone:
Chronic elevation of growth hormone levels in humans usually results in either gigantism or acromegaly. GH, besides affecting skeletal growth, can also influence other organ systems, in particular, the liver and kidney. In the kidney, it has been associated with glomerulosclerosis and nephropathy. In the liver, it has been shown to cause an increase in liver size, as a consequence of both hyperplasia and hepatocyte hypertrophy. The hepatocellular lesions associated with high GH levels progress with age. See Quaife, et al, Endocrinol., 124: 49 (1989).
There is reason to believe that excessive GH activity in the liver is deleterious to health. Mice that express GH transgenes typically live to only about one year of age, while the normal life expectancy for mice is 2–2.5 years. A major cause of death in the GH transgenic mice has been liver disease.
Growth hormone (GH) is an essential regulator of carbohydrate and lipid metabolism, participating in glucose uptake and usage, accelerating fat expenditure, preventing triglyceride accumulation, and facilitating lipid mobilization in adipose tissues. Growth patterns and body compositions of transgenic mice expressing GH analogs have been characterized in our laboratory (Knapp et al. 1994). One transgenic mouse line expresses a GH antagonist (GHA) and is dwarf. As these mice age, they become obese.
Chronic depression of GH levels can also impair health.
Growth Hormone Antagonists:
In view of the foregoing, it has been suggested that if a subject is suffering from excessive GH activity, it can be useful to inhibit such activity by inhibiting the production, release or action of GH, or facilitating the elimination of GH.
Among the agents useful for this purpose are those which are competitive binding antagonists of GH. It was discovered that certain mutants of GH are useful for this purpose. Kopchick, U.S. Pat. No. 5,350,836.
In order to determine whether it is appropriate to initiate or terminate use GH antagonists or other GH-inhibiting drugs, it is important to be able to monitor GH activity.
Monitoring of GH Activity:
The most straightforward marker of GH activity is the serum level of GH per se. For humans, the mean GH concentration (ug/L) in blood is
preadolescent4.6early adolescent4.8late adolescent13.8adult1.8ISS (10 y old)3.5GH deficient1.4IDDM (boys)9.0Obese (male)0.66(lower than controls)Fasting6.7(higher than controls)Hyperthyroid1.9(higher than controls)ISS = idiopathic short stature,IDDM = insulin dependent diabetes mellitusSee Harvey (1995), supra.
While there is definitely a correlation between high levels of GH in serum, and high levels of GH activity, it must be recognized that both the total number of GH receptors, and the distribution of those receptors among the various organs, will vary from individual to individual. Hence, in determining whether an individual is suffering from excessive GH activity, and prone to develop adverse clinical sequelae, it is helpful to identify a metabolite which is produced or released in direct or indirect response to GH and, in particular, one which is substantially liver-specific so that the specific threat to liver function can be assessed.
Another marker of GH activity is insulin-like growth factor-1 (IGF-1). IGF-1 is a 70 amino acid single chain protein, with some structural similarity to proinsulin, which is closely regulated by GH secretion. While the majority of IGF-1 synthesis occurs in the liver, many other tissues, including bone and skeletal muscle, also release IGF-1 in response to GH. IGF-1 levels have been used by clinicians to confirm suspected cases of acromegaly. However, it would be desirable to have a marker, or combination of markers, which was more liver specific than IGF-1, for use in monitoring and predicting the effect of chronic elevation of GH levels on liver function.